Description of new species of Trematoda from bats of Southeastern Mexico and a new classification for Brachylecithum rileyi n. comb. (Dicrocoeliidae)

To date, 23 species of trematodes have been reported in bats from Mexico. However, in some regions of Mexico, such as the Yucatan Peninsula, many species of bats do not have helminthological records. Here, we sampled bats in four localities in Southeastern Mexico from April 2017 to February 2022. Parasites were collected from the intestine of four species of bats: Pteronotus fulvus, Eumops nanus, Noctilio leporinus and Nyctinomops laticaudatus. Conventional morphological techniques and molecular tools with the 28S ribosomal gene were used to describe the helminths. We discovered that our parasites represent three new species and a new combination of trematodes. We found that the new species have morphological differences with their congeneric species, and we complement this information whit molecular data. Furthermore, we found morphological and molecular evidence that places Dicrocoelium rileyi within the genus Brachylecithum. This study points out the importance of comparing morphological and phylogenetic information. Supplementary Information The online version contains supplementary material available at 10.1007/s11230-023-10127-y.


Introduction
In Mexico, more than 50 species of helminths have been recorded in bats, of which 23 are trematodes (Caspeta-Mandujano et al., 2017;Panti-May et al., 2021;Moguel-Chin et al., 2023) Probably in this group of mammals it is still possible to find a wide variety of new species of trematodes (e. g.Fernandes et al., 2019;Cacique et al., 2023), because the records are based on morphology (Salinas-Ramos et al., 2017).However, the use of molecular techniques and phylogenetic analyzes have proven to be powerful methods to detect new species and separate species that are difficult to differentiate with morphological characters (Hernández-Mena et al., 2022).Particularly, in regions such as the Yucatan Peninsula, many species of bats do not have helminthological records and most of them have been reported recently (Moguel-Chin et al., 2023).In previous studies (Panti-May et al., 2021;Moguel-Chin et al., 2023), where the helminthic fauna of several species of bats from the Yucatan Peninsula was recorded through morphological and molecular analysis with DNA sequences, two new species of Limatulum Travassos, 1921 and one new species of Pygidiopsis Looss, 1907 were found, but they were not described due to the objectives of those investigations.Furthermore, in Moguel-Chin et al. (2023) Dicrocoelium rileyi Macy, 1931 was also recorded, a species that, based on morphological observations, has been suggested to belong to Brachylecithum Shtrom, 1940.To date, eight species have been described for the genus Limatulum, and only five have been recorded from Mexico in six different families of bats (Phyllostomidae, Vespertillionidea, Emballonuridea, Molossidae, Natalidae and Mormopidae): L. aberrans Caballero and Bravo, 1950;L. diminutum Chandler, 1938;L. gastroides Macy, 1935;L. limatulum Braun, 1900, and L. oklahomense Macy, 1931(Salinas-Ramos et al., 2017;Caspeta-Mandujano et al., 2017).On the other hand, the genus Pygidiopsis includes 14 species (Sohn et al., 2016) but only P. macrostumum Looss, 1907 has been reported in Mexico (Panti-May et al., 2021;Moguel-Chin et al., 2023).However, Moguel-Chin et al. (2023) observed that some morphological characters of the specimens recorded in Mexico differ from the specimens of P. macrostumum described by Simões et al. (2005).In addition, the species D. rileyi had also already been recorded in the country (e.g.Caspeta-Mandujano et al., 2017;Martínez-Salazar et al., 2020).Therefore, the objectives of this study are to describe three new species of trematodes, two from Limatulum and one from Pygidiopsis, propose a formal taxonomic change from D. rileyi to Brachylecithum and explore the phylogenetic relationship of these helminths with DNA sequences.

Specimen collection
In the Yucatan Peninsula, the bats Eumops nanus (Miller), Nyctinomops laticaudatus (Geoffroy), Pteronotus fulvus (Thomas) and Noctilio leporinus (Linnaeus) were collected in three sites from May 2017 to February 2021, and in Veracruz P. fulvus was collected in April 2017, under permits from the Mexican Ministry of Environment (SGPA/DGVS/03705/17 and SGPA/DGVS/00786/21; FAUT-0170 and FAUT-0056 respectively).Bats were captured using mist nets (12 m wide × 2.5 m high) for one night, placed in cloth bags, and identified following Medellín et al. (2008).Bats were anesthetized with isoflurane and euthanized by overdose of sodium pentobarbital.The heart, lungs, stomach, liver, small and large intestines, and mesenteries of each specimen were collected and stored in 96% ethanol.All collected organs were dissected from each bat and immersed in distilled water in Petri dishes using a stereo microscope (Olympus SZ2-ILST).Helminths were collected, counted and preserved in 70% ethanol until morphological and molecular identification.Unlike the helminths collected in Yucatan, the helminths in Veracruz were collected immediately after sacrificing and dissecting the bats, so the trematodes were still alive and were killed with 4% hot formaldehyde for morphological study and others were placed directly in absolute ethanol for molecular study.Bats from the Yucatan Peninsula were deposited in the Colección Zoológica (CZ), Campus de Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán (Supplementary 1), while bats from Veracruz were deposited in the Colección de Mamíferos del Museo de Zoología "Alfonso L. Herrera", Facultad de Ciencias, Universidad Nacional Autónoma de Mexico.

Morphological analysis
All the trematodes destined for the morphological study were stained with carmine acid or Gomori trichrome, dehydrated through an ethanol series, cleared Page 3 of 17 4 Vol.: (0123456789) in methyl salicylate, and permanently mounted in Canada balsam.Specimens were studied under light microscopy (Leica DM500).The measurements are presented in micrometres (μm) with the range followed by the mean in parentheses.Trematodes were identified to genus level using the taxonomic key of Bray et al. (2008).For identification and comparison at the species level, the original descriptions of the respective articles were used.Specimens were deposited in the Colección Nacional de Helmintos (CNHE), Universidad Nacional Autónoma de Mexico.

DNA sequencing and phylogenetic analyses
The procedures for extraction, amplification and DNA sequencing of trematodes were detailed in a previous study (Moguel-Chin et al., 2023).We amplified the 28S gene of ribosomal DNA with the forward primer 391 5′ -AGC GGA GGA AAA GAA ACT AA-3′ (Stock et al., 2001) and the reverse primer 536 5′ -CAG CTA TCC TGA GGG AAA C-3′ (Stock et al., 2001) which amplify a fragment of approximately 1200 base pairs (bp).The obtained sequences were deposited in the Genbank.For the phylogenetic analyses, the new sequences were aligned with those of sequenced species that are available in the Genbank (access numbers in the trees) (Supplementary 2).The phylogenetic method used was Maximum Likelihood (ML) and was executed with 1,000 Bootstrap repetitions to obtain support values for the clades.The phylogenetic analysis procedure is also described in greater detail in Moguel-Chin et al. (2023).Life Science Identifier: 94FCC493-C98B-4E12-B3FC-183F988856A0 Etymology: The specific name of the new species refers to the name of the species of bat that is the host of this parasite.

Remarks
Limatulum fulvum n. sp.conforms to the diagnosis of Limatulum as given by Travassos (1921) by presenting some characteristics such as tegument spinous, testes at or near the level ventral sucker, ovary anterior to the testes and vitellarium in the forebody.The new species is characterized by having the smallest cirrus sac of all species of the genus (Table 1).This new species is also characterized by having a long oesophagus, a character shared with L. mcdanieli Brooks &Coggins 1983 andL. aberrans Caballero &Bravo-Hollis, 1950 but that differentiates it from the rest of the species.Limatulum fulvum n. sp.can be readily distinguished from L. mcdanieli by having smaller oesophagus (24.4-75 vs. 86-111); in the new species the vitelline glands are confluent whereas in L. mcdanieli the vitelline glands are in two clusters; finally, L. mcdanieli differs from L. fulvum n. sp. by exhibiting an anterior fleshy lobe of tissue on the ventral sucker.The new species has an oesophagus similar in size to L. aberrans, but differs from this species by presenting a smaller ovary and testes (Table 1); the vitellarium in L. fulvum n. sp.does not reach the pharynx, while in L. aberrans does reach the level of the pharynx; finally, the anterior extension of the uterus in L. fulvum n. sp. is up to the anterior region of the testes, while in L. aberrans it exceeds the anterior region of said gonads.Other characters that differentiate the new species from its congeners are: the vitelline glands are confluent in L. fulvum n. sp.while in all the others (with the exception of L. aberrans and L. diminutum Chandler, 1938) they are not confluent and forms two lateral clusters; the vitellarium does not reach the level of the pharynx in the new species, while in all the others (with the exception of L. mcdanieli) it does reach the pharynx or the oral sucker; finally, the testes are distributed posteriorly to the ventral sucker, while in L. diminutum, L. gastroides Macy, 1935, L. limatulum Travassos, 1921, L. ocklahomensi Macy, 1931 and L. nanum n. sp.they are at the level of this sucker.

Remarks
Limatulum nanum n. sp.conforms to the diagnosis of Limatulum as given by Travassos (1921) as mentioned above.This new species can be differentiated from its congeners by the size of the cirrus sac: it is larger than the sac of L. fulvum n. sp., but smaller than the rest of the species (Table 1).The new species can be easily differentiated from L. aberrans, The new species can be distinguished from L. limatulum because in L. nanum n. sp.all the tegument is covered by spines and the vitelline glands reach the posterior edge of the ventral sucker whereas in L. limatulum the spines extend from the anterior border to the testes and the vitelline glands reach the middle of the ventral sucker.Limatulum nanum n. sp.differs from L. oklahomense because in the new species the genital pore is immediately to one side of the ventral sucker and in the middle of this sucker, while in L. oklahomense it is lateral to the testis and is posterior to the ventral sucker.Finally, L. nanum n. sp. is also different from L. gastroides, because the new species is larger in length and has narrower eggs than L. gastroides.

Remarks
Brachylecithum rileyi n. comb. is a species of Dicrocoeliidae that was originally described as Dicrocoelium rileyi by Macy (1931) who found it parasitizing the intestine of Tadarida brasiliensis (Le Conte) in Kansas, USA.Since then, it has been recorded in several places in Mexico in the same species of bat (Caballero and Caballero 1969;Guzmán-Cornejo et al., 2003;Caspeta-Mandujano et al., 2017;Falcón-Ordaz et al., 2019) and in N. laticaudatus (Moguel-Chin et al., 2023).When the species was described, the genus Brachylecithum had not yet been erected, which was formally described by Shtrom (1940).This author considered that the extension of the caeca and of the vitellarium were important characteristics to separate Brachylecithum from Dicrocoelium, because in Brachylecithum the caeca are short (terminate far from posterior extremity of body) and the vitellarium is composed of few large follicles and does not reach anteriorly to the ovary, while in Dicrocoelium the caeca are longer (reaching closer to the posterior extremity of the body) and the vitellarium is composed of small follicles that do reach the ovary and even in some species, the follicles extend beyond the anterior border (Pojmańska 2008).Therefore, due to the extension of the caeca and of the vitellarium in B. rileyi n. comb., this species agrees rather with the diagnosis of Brachylecithum and not with Dicrocoelium.Unfortunately, in more recent studies, where it can also be seen that the morphology of the specimens agrees with Brachylecithum (i.g.Caspeta- Mandujano et al. 2017;Falcón-Ordaz et al. 2019), the researchers did not note the diagnostic differences between these genera, and they continued to name their specimens as D. rileyi.

Phylogenetic relationships and genetic distance
The aligned data set for the 28S gene was of 1446 base pairs (bp) long and consisted of 69 sequences of Trematoda including sequences of Brachylecithum rileyi n. comb., Pygidiopsis noctilus n. sp., Limatulum fulvum n. sp. and Limatulum nanum n. sp.from Yucatan, Campeche and Veracruz, Mexico.The substitution model selected for this data set used to infer the ML phylogenetic hypothesis was GTR+CAT.The nucleotide frequencies were A= 0.215 C= 0.221 G= 0.318 T= 0.246.The ML tree had a value of ln = -13616.961705.Our specimens were grouped in three main clades (Fig. 7).The first clade named "A" grouped species of Lecithodendriidae, Stomylotrematidae, Prosthogonimidae, Pleurogenidae, Phaneropsolidae and Microphallidae (superfamily: Microphalloidea) (bootstrap = 100).Our new species of Limatulum were grouped in the same subclade as a sister group of Pleurogenidae (Boot-strap= 100).Our sequences of L. fulvum n. sp.from Yucatan and Veracruz presented low genetic differences of 0.09%.This species presented genetic differences of 3.8-4.1% with L. nanum n. sp.In the second clade named "B", species of Heterophyidae (superfamily: Opisthorchioidea) were grouped with high support values (Bootstrap= 100) and the specimens of P. noctilus n. sp. were nested as a sister species of P. macrostomum (Bootstrap= 100).The sequences of P. noctilus n. sp.presented a null intraspecific genetic difference, but the genetic variation with the sequences of P. macrostomum from Brazil were of 0.5-0.6%.The third clade named "C" comprised members of the family Dicrocoeliidae (superfamily: Gorgoderoidea).In this clade, Page 13 of 17 4 Vol.: (0123456789) Brachylecithum was not monophyletic, and three subclades of this genus were obtained.Particularly, our sequence of B. rileyi n. comb.was grouped as sister species of Brachylecithum grummti Odening, 1963 from a bird of Brazil (bootstrap = 100).The genetic distance between these sister species was 2.1%.

Discussion
The new Limatulum species described in this study are the ninth and tenth described for this genus in the Americas.On one hand, L. fulvum n. sp. is readily distinguishable from most congeners mainly by having: the smallest cirrus sac of all species in the genus, a long oesophagus, confluent vitelline glands that do not reach the level of the pharynx, and testes posterior to the ventral sucker.On the other hand, L. nanum n. sp.can be distinguishable from the other congeneric species because does not have a visible oesophagus, it has non-confluent vitelline glands in two lateral clusters and it has testes at the level of ventral sucker, in addition to the combination of several characteristics such as: the position of the genital pore, the size of the cirrus sac and the size of the eggs.These last characteristics are used for other authors to separate the species from the congeneric species (Brooks and Coggins 1983;Lunaschi et al., 2003).The 28S sequences of the new species represent the first DNA sequences for the genus Limatulum, which allowed us to explore the phylogenetic position of the genus.Phylogenetically, the new species are nested among themselves in a clade independent from other families of Microphalloidea.This result is interesting because Limatulum has been associated as part of the Phaneropsolidae (Lotz y Font 2008); however, in our phylogeny, Phaneropsolus is not grouped with Limatulum, indicating that both belong to different families.Limatulum has also been associated with Lecithodendriidae, but phylogenetically it does not group with species of this family either.However, the Limatulum clade is resolved as the sister group of the Pleurogenidae clade sensu Tkach et al. (2019).Information on the phylogenetic position of Limatulum indicates that an eventual detailed taxonomic review of the diagnoses of Phaneropsolidae, Lecithodendriidae and Pleurogenidae is necessary, since the limits between these families seem to be diffuse, which has led to confusion and movements within their classifications.
Pygidiopsis noctilus n. sp. is the first species of the genus described in Mexico and the fifteenth species of the genus.Natural infections of this genus in mammals are rare.This group of parasites are generally found in birds and fishes, and the few records in mammals (e.g.rodents) come from experimental infections (Ostrowski de Núñez 1995; Ostrowski de Núñez 1996; Sohn et al. 2016).Pygidiopsis noctilus n. sp. is easily distinguished from most congeners because it possesses vitelline glands that reach the posterior border of the ventral sucker and the uterus extends to the pharynx.These characteristics are mentioned by Ostrowski de Núnez (1995) to differentiate the species of the genus.However, P. noctilus n. sp.can also be diagnosed by the combination of some characters such as: the ventral / oral sucker length ratio, the size of eggs, ovary and testes.For Pygidiopsis, we found sequences only for one congeneric species, and the tree show that P. noctilus n. sp. and P. macrostomum are sister species.Pygidiopsis macrostomum was recorded in Brazil through experimental infections of Rattus norvegicus (Berkenhout) (Simões et al. 2005) and also Pygidiopsis sp.(cf.macrostomum) was found in Cuba in N. leporinus (the same host of the new species) (Odening 1969).In the databases we only found DNA sequences of Pygidiopsis macrostomum from Brazil, but not from Cuba.Due to the geographic distribution, the host affinity and the similarity of some diagnostic characteristics such as the size of the eggs (18-19 × 9-11 vs 18-20 × 9 -10 in P. noctilus n. sp.) of the Pygidiopsis from Cuba, we infer that it is P. noctilus n. sp.instead of P. macrostomum, but to corroborate it will be necessary to sequence the specimens from other geographical regions in the future.The few genetic distances between P. noctilus n. sp. and P. macrostomum may indicate a recent divergence between both species and a low substitution rate of the 28S region, which is why there are also some similarities in morphology.However, the geographic distribution, the phylogenetic grouping and the specific morphological differences between both species are evidence to confirm the existence of the new species.
Regarding Brachylecithum, only some species of this genus have been reported in mammals (Rodentia, Insectivora and Chyroptera) (Casanova y Ribas 2004).The new combination of Brachylecithum rileyi increases the number of records for the genus in mammals.When the morphology of this species is studied it may be obvious that it does not belong to Dicrocoelium sensu stricto, but we think that several factors could have influence the delay in recognizing B. rileyi n. comb., and we can mention at least two.On one hand, the species was described before Brachylecithum was erected, and because the original description was published in Russian, information did not flow so quickly to the Western world and consequently it continued to be erroneously named D. rileyi for all these years in a domino effect.On the other hand, the uterus may become saturated with eggs causing that the extension of the caeca and vitelline follicles could not be assessed successfully, and this could have confused the determination of the specimens at the genus level in previous works.In Page 15 of 17 4 Vol.: (0123456789) the clade "C" Brachylecithum form several subclades.The phylogeny of the family are poorly resolved and the relation between their genera were unclear, so some authors have suggested a systematic review of several genera of the family Dicrocoeliidae which may explain the non-monophyly of the genera (Tkach et al., 2001a(Tkach et al., , 2001b)).
The identification bases only in the morphology may allow inaccurate identifications due to the natural variations in size, organ position and structure body shape according to intrinsic parasite factors (e.g.age) and the methods of collection and fixation (Aldhoun et al., 2018).This study points out the importance of comparing morphological and phylogenetic information.It is essencial to continue with the studies on parasites of bats, especially on members of families that lack records of helminths.

Fig. 7
Fig. 7 Phylogenetic tree based on the Maximum Likelihood analysis constructed on partial large subunit ribosomal gene (28S) of Trematoda species from different hosts (likelihood =

Table 1
Morphometric measurements of the species of the genus Limatulum described in this study NA: not available *No measurements were presented in the description **In the original description there are no official measurements of the structure, but this measurement was calculated thanks to the scale and the diagram Vol:. (1234567890)

Table 2
Morphometric measurements of the species of the genus Pygidiopsis described in this study